PROJECT SUMMARY Genetic variation in humans contributes to an individual's likelihood to develop an alcohol use disorder. The genetic variation that influences alcohol abuse liability is therefore an important target for study, but it has been difficult to identify specific liability genes. Laboratory studies in animal models have been extremely useful in elucidating the molecular pharmacology of alcohol (ethanol), but laboratory derived genetic manipulations rarely model the naturally occurring genetic variation that is observed in wild populations. As such, predicting relevant human allelic variation has been difficult. Here, we study the natural allelic variation found in wild strains of the the nematode worm Caenorhabditis elegans to identify alleles that are tolerated in the wild and can modulate the function of pathways that impact physiological responses to ethanol. C. elegans is an important model species with demonstrated relevance to humans; there is striking conservation between the machinery of nervous system function in worms and humans, and genes that influence ethanol response behaviors in worms also influence the likelihood to develop alcohol use disorders in humans. This collaborative proposal brings together the diverse expertise of two laboratories. We take advantage of a unique resource, recombinant inbred lines (RILs) derived from four genetically diverse wild strains to identify natural allelic variation that can modulate the effects of ethanol. We have shown that the parent wild-type strains and the derived RILs display a range of phenotypes in different behavioral responses ethanol. We will exploit the efficiency and ease of manipulating the C. elegans model to carry out high throughput analyses that will identify genetic variation that alters behavioral and/or transcriptional responses to ethanol. We will directly test the causal nature of candidate ethanol response allelic variants, identified by quantitative trait locus mapping, through the use of gene editing techniques (CRISPR-Cas9) to introduce the allele into strains that carry different alleles. The ability to compare and contrast the influence of genetic variants on different responses to ethanol brings further power to our analyses. We will identify the genes that impact the physiological responses in each behavior uniquely, and second, we will identify genes that affect the behavioral responses across ethanol response phenotypes. We will also assess the impact of alleles that modulate transcription in response to ethanol on behavioral responses. These different analyses can inform us of the molecular mechanisms underlying these different responses to ethanol. Together, these studies will provide both specific and more general novel insights into the neurogenetics of ethanol. We will establish the degree to which genetic variation in known or novel biological pathways that mediate or modulate the effect of ethanol can change responses to ethanol and the degree to which variation in those genes is tolerated in the wild. These data will inform our understanding of human liability to abuse alcohol.